Detection of Mycobacterium species of the Mycobacterium avium complex (MAC) in clinical samples is important as a diagnostic tool. M. avium complex organisms include M. avium, M. intracellulare and other species that are difficult to differentiate from these, such as M. paratuberculosis. MAC organisms are frequently found in clinical samples and are common causative agents of opportunistic infections in immunocompromised individuals, such as HIV-infected individuals or individuals undergoing chemotheraphy or using immunosuppressive drugs (Good et al., 1982, J. Infect. Dis. 146: 829-833; Gill et al., 1985, J. Clin. Microbiol. 22: 543-546). Therefore, assays that can detect MAC species and distinguish them from other species are important for clinical diagnosis.
Clinical diagnostic assays for Mycobacterium species often rely on time-consuming methods that analyze bacterial physical characteristics (e.g., staining and microscopic detection), physiological characteristics (e.g., growth on defined media) and/or biochemical characteristics (e.g., membrane lipid composition). Such methods often require relatively high bacterial concentrations in the sample and may require a high degree of experience and expertise to properly determine the infective species. Diagnostic assays that require in vitro growth of the bacteria are costly both in terms of delayed or inappropriate early treatment of the patient and in terms of the amount of laboratory equipment and space required to culture Mycobacterium, which is often difficult to grow in vitro.
Assays that use molecular biology techniques to detect the presence Mycobacterium nucleic acid in the sample have been introduced to increase the sensitivity and relative speed of diagnosis (U.S. Pat. Nos. 5,030,557, 5,567,587, 5,595,874, 5,601,984 and 5,677,128; PCT No. WO95106755). These assays may directly detect the nucleic acid sequences present in the sample or may rely on in vitro nucleic acid amplification of nucleic acids present in the sample before the detection step (U.S. Pat. Nos. 5,554,516, 5,766,849, 5,906,917, 5,908,744; European Patent Nos. EP 0528306 and EP 0818465; and PCT Nos. WO 9636733 and WO 9723618). Many in vitro nucleic acid amplification reactions require amplification oligonucleotides that serve as primers for a polymerase reaction that uses the nucleic acid present in the sample as a template. Detection of the amplified nucleic acid often requires use of specific nucleic acid probes that hybridize to the amplified sequences to produce a detectable signal or complex.
The present invention provides compositions and in vitro nucleic acid amplification methods that produce relatively long amplified nucleic acid sequences to allow detection of MAC species present in a biological sample.